Plant materials
Panax ginseng is Korean wild ginseng. Wild ginseng grows naturally within its natural
habitat conditions (Figure 2). Adventitious roots were induced and proliferated from wild Panax ginseng root, and cultured in MS medium supplemented with NAA and IAA.
Figure 2. Korean wild ginseng (Panax ginseng Meyer).
Establishment of adventitious root cultures Thiamine Hydrochloride (2.5), Glycine (2). Supplemented with 10.87μmol Naphthalene acetic acid (NAA), 1.43μmol Indole-3-acetic acid (IAA) and 3% sucrose. The pH of the medium was adjusted to 6.0 before autoclaving at 121℃ and 1.2 Kgf/cm2 pressure for 15 min.
Petridish culture condition
Adventitious roots were cut into 10 pieces, each 1-2 cm and cultured in petridish (10cm in diameter and 1.5 cm in height) with lid containing 50 mL MS solid medium was sealed with a wrap (Advantec, USA) and cultured at 23 ± 2℃ under dark condition. Adventitious roots were sub-cultured every 30 days.
Flask culture condition
Fresh roots (0.8g) were inoculated into a 100 mL erlenmeyer flask containing 50 mL MS liquid medium with 10.87 μmol NAA, 1.43 μmol IAA and 5% sucrose. Cultures were shaken at 110 rpm in the light at 23 ± 2℃ for 30 days.
Bioreactor culture condition
The bioreactors were maintained at 23 ± 2℃ in a light condition until harvest.
Adventitious roots were sub-cultured every 30 days in 15L bioreactor (Biopia, Korea)
containing 5L MS medium. Forty-five g fresh weight of adventitious roots was inoculated into bioreactor for proliferation. After 30 days, the proliferated adventitious roots were used as explants for further experiments. The culture system showed in figure 3.
Figure 3. Culture system of Panax ginseng. (A) Root of Panax ginseng; (B) Explant of ginseng root; (C) Adventitious root proliferation; (D) Adventitious root culture in flask; (E) Adventitious root culture in bioreactor; (F) Harvest.
Gamma irradiation
Adventitious roots (1-2cm) were placed in plastic petridishes (10 pieces adventitious roots per pertridish), grown at 23 and cultured ℃ for 5 days in MS medium with 10.87 μmol NAA, 1.43 μmol IAA and 3% sucrose. They were exposed to gamma radiation from cobalt (60Co) source using a γ-radiation apparatus at the Applied Radiological Science Research Institute, Jeju National University. Irradiation dosages were 0 (non-irradiated), 25, 50, 75, 100 and 125Gy. For each dosage, 3 petridishes of the samples were exposed in triplicate.
Effects of gamma irradiation on survival rate of adventitious roots were evaluated by measuring the number of survival adventitious roots after 5 weeks culture.
Selection of cell lines from suspension culture
Secondary roots of survival main roots were transferred into 50 mL liquid MS medium with NAA and IAA in flask. Cell line was selected according to the secondary roots number, length, diameter, growth ratio.
Determination of root weight and growth ratio
Fresh weight (FW) and dry weight (DW) were measured after 30 days growth in flask.
FW and DW of roots were determined as follows. Roots were separated from the medium by passing through a 1 mm stainless steel sieve. Root FW was measured after rinsing once with tap water and blotting away surface water and root DW was recorded after roots were dried to a constant weight at 38℃ for several days (Kim et al. 2004). Root growth ratio was
calculated by using the following formula (Yu et al. 2002):
Extraction of crude saponin
Extraction and determination of ginsenosides were carried out by modifying the method of Kwon et al. (2003). Ultrasound-assisted extraction was performed with an ultrasonic water bath (Branson ultrasonics, USA). The output power is 117 volts and the frequency is 50/60Hz. Sample powder 0.5 g was placed into a 100 mL conical flask, into which 30 mL of 80% (v/v) methanol–water were added. Then the flask was sonicated for 1 h in an ultrasonic water bath. The extract obtained was evaporated using a rotary evaporator under vacuum at 55℃. The evaporated residue (total extract yield) was dissolved in 20 mL of distilled water and washed twice with 20 mL of diethyl ether to remove the fat contents using a separatory funnel. The aqueous layer was extracted four times with 20 mL of water-saturated n-butanol.
The butanol solution was washed twice with 30 mL of distilled water to remove the impurities, thereby obtaining crude saponins. The remaining butanolic solution was transferred to the tarred round bottom flask for the evaporation using a rotary evaporator
Figure 4. Extraction procedures of crude saponin in Panax ginseng.
Determination of ginsenoside content by HPLC
The HPLC conditions for ginsenoside assay was slightly modified the previous report (Park et al. 2007). Quantitative determinations were achieved by HPLC using a Capcell-pak C18 MG (4.6 × 250 mm) column (Shiseido, Japan), Waters 2998 Photodiode Array Detector, Waters 2690 Separations Module and Empower Program (Table 1).
Table 1. HPLC conditions for ginsenosides analysis.
Mobile phase Distilled water and Acetonitrile
Flow rate 1 mL/min
The solvents of HPLC grade were used. The water used in this study was treated with a Milli-Q water purification system (Millipore, USA). The mobile phase of HPLC gradient was conducted as follows (Table 2). Ginsenosides Rg1, Re, Rf, Rh1, Rb1, Rc, Rb2 and Rd standards were purchased from BTGin Co., Ltd (Daejeon, Korea). Digoxin was used as internal standard.
Table 2. Mobile phase of HPLC gradient condition for ginsenosides analysis. Solvent (A): Distilled water, Solvent (B): Acetonitrile.
Stock solutions for the 4 ginsenosides were prepared separately in 100% MeOH. Digoxin stock solution was prepared in 70% MeOH. Working solutions were prepared in methanol by mixing known amount of all the ginsenosides together. Five concentrations were made for standard curves, each concentration was 60, 120, 240, 320, 480 ppm. Ginsenosides were detected at a wavelength of 203 nm with the peak areas corresponding to ginsenosides from the samples matching retention times as authentic ginsenoside standards.
Analysis of ginsenosides contents was performed according to Son et al. (1999a) and Yu et al. (2000). The total ginsenoside content was calculated as the sum of individual ginsenoside fractions.
The ginsenoside content of ginseng adventitious roots was calculated as:
(GC: ginsenoside content; SGC: sample ginsenoside concentration from HPLC; SV: sample volume; AR: adventitious root)
Method validation and statistical analysis
Stock solutions for the 8 ginsenosides were prepared separately in 100% methanol.
Digoxin stock solution was prepared in 80% methanol. Working solutions were prepared in 100% methanol by mixing known amounts of all the compounds. The linear range, limit of detection (LOD) and limit of quantification (LOQ) were studied for the developed method.
The linearity of calibration curve was tested by standard analysis. The calibration curves of individual ginsenosides were constructed using a range of five concentrations of the standard, and LOD and LOQ for each analyte were evaluated at signal-to-noise ratios (S/N) of 3:1 and 10:1, respectively.
Statistical analysis was performed according to the SPSS system. Mean and standard errors were used throughout and statistical significance between the mean values was assessed by applying a Duncan’s multiple range tests. A probability of P < 0.05 was considered significant.
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